Abstract
Purpose: :
The tetracycline inducible expression tet-ON system requires two elements, the rtTA (reverse tetracycline-controlled transactivator) and the expression cassette containing TRE (tetracycline response element) combined with a mini CMV promoter (PminiCMV) and the gene of interest (GOI). This is achieved by two separate constructs, one contains the rtTA, and the other has TRE-FOI. Here we describe a one-piece construct to control the expression of rhodopsin and the P23H mutation.
Methods: :
The one-piece vector pLT1P4 was based on pLenti4 (Invitrogen). The backbone of the vector was constructed by releasing the tet-inducing element from pLenti4. A CMV promoter and rtTA-m2 fragment was excised from a pre-constructed plasmid, pcDNA3.1(+)-rtTA-m2 and was then inserted into the pLenti4 backbone to generate pLT-rtTA-m2. The EcoRV fragment of pLenti4, which contains the Gateway recipient sequences attR1 and attR2, was inserted into pLT-rtTA-m2, down stream of CMV-rtTA-m2 with the same orientation to complete the construction of pLT1P4. The introduction of the Gateway recipient sequences facilitate the subsequence cloning of GOI. The 3’LTR/SV40 polyadenylation signal of pLenti4 serves as polyA signals for both rtTA-m2 and GOI. A pre-constructed TRE-miniCMV promoter with the cDNA of bovine rhodopsin fused to GFP (Moritz et al, JBC, 2001) was subcloned into pENTR2b (Invitrogen) to generate pENTR-TRE-Rho-GFP. An LR recombination reaction between pLT1P4 and pENTR-TRE-Rho-GFP using a Clonase II mix (Invitrogen) converted the TRE-Rho fragment into pLT1P4, resulting in pLT1P4-Rho-GFP. After transfection to human 293 cells, doxycycline (Dox, 2 µg/ml) was added to the culture medium. Cells were harvested 2 days later and examined by immunoblot analysis or fluorescence microscopy. Rhodopsin mutation P23H was generated using the Qick-Change kit (Stratagene), to construct pLT1P4-Rho-GFP-P23H.
Results: :
Immunoblot analysis showed a dramatic induction of rhodopsin-GFP expression in pLT1P4-Rho-GFP transfected cells treated with Dox. The expression was undetectable in control cells with or without Dox as well as in cells transfected with pLT1P4 without Dox treatment. Strong GFP signal was seen in the membrane of transfected cells treated with Dox.
Conclusions: :
This construct simplifies the original binary Tet-On system and is effective in induce expression of GOI with minimum leak. Using a rhodopsin promoter to drive the rtTA expression, it can be used to control expression of rhodopsin-GFP fusion protein and its mutants in rods in transgenic animals.
Keywords: photoreceptors • gene/expression